Transcript Slide 1

PCB6528 Spring 2014 - Exam 2 – Chase
Name __KEY__________________________
The exam was written based on 100 points. Dr. Cline will weight its contribution to your final grade
based on the fact that the exam covers material presented in 6 classes.
Important – please keep your answers short; confine your answers to the space provided; do not
write on the back of any pages !
1 (24 pt) The following statements relate to our class discussion of organelle genomes. For each
statement, indicate whether the statement applies to the plastid genome (pt), the plant
mitochondrial genome (mit), both plastid and plant mitochondrial genomes (both), or neither of
these genomes (none). I was strict on these – right or wrong, no part credit
Circular maps based upon DNA sequencing both
Linear and circular molecules of various sizes visualized by FISH or EM both
Subgenomic forms mediated by recombination through direct repeats mit
Inversion isomers mediated by recombination through inverted repeats both
Gene order is generally conserved among plant species pt
Gene order is highly variable among plant species mit
Gene coding content varies among plant species both
Genome can be genetically transformed through homologous recombination pt
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PCB6528 Spring 2014 - Exam 2 – Chase
Name ____________________________
2 (24 pt) In our discussion paper we saw that plastid genome transformation could be used to
genetically mark plastid genomes so that we could follow their fate in grafting experiments. In the
experiment described below, genetically marked plastids were used to monitor the transfer of
plastid DNA to the nucleus. Explain why this experiment estimates only the frequency of nonfunctional DNA transfer from plastid to nucleus, rather than the frequency of a plastid gene
undergoing a transfer such that its gene function is re-established from the nucleus.
(8 pt) In this experiment, the neo gene in the plastid was already fused to a nuclear promoter, so it
had only to move to the nucleus to be expressed
In a functional gene transfer from plastid to nucleus, the transferred gene would need to acquire a
nuclear promoter (10 pt) and also an in-frame targeting signal to direct the protein product back to
the plastid (6 pt)
Modified from Timmis et al. Nat Rev Genet 5:123
A construct that consists of chloroplast sequences (C and D) that
flank two selectable marker genes is inserted into the chloroplast
genome through homologous recombination, thereby
transforming the native plastome into a TRANSPLASTOME (a). One
of the selectable genes (aadA) is designed for exclusive expression
in the chloroplast and incorporation of this marker confers
spectinomycin resistance. The other gene, a neomycin
phosphotransferase gene neoSTLS2 , includes a nuclear promoter
and contains a nuclear intron within the reading frame. NeoSTLS2
confers kanamycin resistance only when it is transposed to the
nucleus. Continuous selection of growing leaf cells on
spectinomycin medium allows transformed plastomes to be
selected and eventually the transplastome entirely replaces the
native chloroplast genome, such that all copies of the chloroplast
genome contain the two selectable marker genes (b). The
transplastomic plants were crossed with wild-type female plants.
Because of strict maternal inheritance of tobacco plastids, progeny
that contained only wildtype chloroplasts were produced.
Selection of progeny seedlings on kanamycin medium allows the
detection of the rare cases in which the neo gene has changed its
location, such that strong expression is promoted from the nuclear
environment (c). Therefore, chloroplast-to-nucleus transposition
must have occurred at some stage during the life cycle of the male
parent of the seedlings that were recovered on kanamycin plates.
The observation that 1 in 16,000 male tobacco gametes contained
a segment of chloroplast DNA newly integrated into the nuclear
genome was unpredictably high.
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PCB6528 Spring 2014 - Exam 2 – Chase
Name ____________________________
3 (21 pt) The following statements relate to our class discussion of organelle gene expression. For
each statement, indicate whether the statement applies to the plastids (pt), the plant mitochondria
(mit), both plastid and plant mitochondria (both), or neither organelle (none). I was strict on these –
right or wrong, no part credit
Genes are transcribed by single-subunit, bacteriophage-type RNA polymerases both
Genes are transcribed by multi-subunit, bacterial-type RNA polymerases pt
Transcription utilizes nuclear-encoded sigma factors pt
Primary transcripts are multi-cistronic operons pt
Genes contain group II introns that undergo self-splicing in the organelle none – trick question, see
the slide regarding the plastid group II introns and the myriad splicing factors required to properly
splice them in the organelle
Site-specific RNA binding proteins of the PPR class are involved in multiple gene expression
processes both
Primary transcripts must undergo C-to-U RNA editing to correct DNA coding errors both
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PCB6528 Spring 2014 - Exam 2 – Chase
Name ____________________________
4 (31 pt) In the two experiments shown below, Schult et al (Plant Cell 19:1329) investigated the
effects of the hcf173 mutation on the thylakoid proteins of Arabidopsis.
What does hcf stand for AND why would the authors expect to see differences in the thylakoid
proteins of the hcf mutant compared to wild-type plants?
(3 pt) High chlorophyll fluorescence
(4 pt) This phenotype results from the inability of the thylakoid- located photosythetic complexes to
utilize harvested light energy (2 pt if you said based on the figure below, but I was looking for a
general explanation
Based on the immunoblotting experiment (below, left): Which thylakoid protein complex appears to
be most strongly affected in this mutant? Explain your reasoning.
(4 pt) PSII
(4 pt) None of the PSII subunits seem to accumulate to even 1/8 wild type levels. PSI proteins also
reduced, but not as much
Based on the in vivo protein synthesis experiment (below, right), which plastid gene/protein appears
to be the one most directly affected by the mutant? Explain your reasoning.
(4 pt) D1 protein
(4 pt) All of the other PSII proteins appear to be translated at wild-type levels, but D1 translation is
much reduced.
Based upon your answers above and our class discussions, what aspect of plastid gene expression
would you next investigate to determine the primary function of the hcf173 gene with respect to
plastid gene expression. Explain your reasoning.
(8 pt) anything that is reasonable. I was thinking any aspect of D1 RNA metabolism (transcript
accumulation, processing, splicing or editing) to see if there is an RNA-based explanation for the
translation phenotype. Some of you focused on protein-protein interactions, or interactions of
hcf173 with other RNAs to see why the other PSII subunits were not accumulating, which was also
fine.
Left - Immunoblot Analysis of
Thylakoid Membrane Proteins
from the Wild Type and the
Mutant hcf173. Twenty
micrograms (WT 1/1 and
hcf173), 10 mg (WT 1/2), 5
mg (WT 1/4), and 2.5 mg (WT
1/8) of protein were
separated by SDS-PAGE and
transferred to a nitrocellulose
membrane. Specific proteins
were immunodecorated
with the indicated antisera.
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Above - In Vivo Protein Synthesis in WildType Plants and hcf173 Mutants. Newly
synthesized proteins of 15-d-old plants
were radiolabeled with 35S-Met for 20
min. (A) Wild-type and hcf173
membrane proteins with equivalent
amounts of radioactivity (100,000 cpm)
or the indicated dilutions were separated
by SDS-PAGE and analyzed by
fluorography.